Cytoplasmic dynein is a high molecular weight, microtubule-based mechanochemical ATPase that is thought to provide a motive force for a number of intracellular motilities. These include membrane-bound organelle transport, endocytotic trafficking, and intracellular organization of structures such as the golgi. Cytoplasmic dynein also localizes to the mitotic spindle and to the kinetochore region of condensed chromosomes where it may play active roles in spindle assembly, position, and chromosome movement during cell division. While in vitro studies and localization data have been suggestive, there is very little direct functional data on what cytoplasmic dynein does in cells. This application describes the initial stages of an in depth analysis of cytoplasmic dynein function. We are proposing here to pursue four distinct but overlapping lines of investigation: (1) a molecular genetic approach to delete or modify the dynein heavy chain sequence in situ (in Dictyostelium) to ask how important these gene products are to cell viability or cytoplasmic organization; (2) an indepth fine structure analysis to correlate the molecular sequence with the visible structural domains of this large polypeptide; (3) a detailed biochemical identification of functional domains such as microtubule-, kinetochore-, and organelle-binding sites; and (4), identification and molecular cloning of polypeptides that interact with the heavy chain, such as regulatory molecules, or polypeptides located on organelles or kinetochores. These four complementary approaches are designed to take maximal advantage of Dictyostelium as an experimental system. The overall goal of this project is to provide understanding of a molecule that potentially participates in a variety of essential cellular functions.